Cartilage tissue engineering on macroporous scaffolds using human tooth germ stem cells

The main objective was to study cartilage regeneration through differentiation of human tooth germ stem cells (HTGSCs) into chondrocytes on different three‐dimensional (3D) scaffolds (PCL, PLLA and PCL–PLLA). Scaffold topographies were studied by scanning electron microscopy and it was found that the scaffolds had interconnected macroporous structures. HTGSCs were isolated from impacted third molar tooth germs of young adult patients and grown for 3 weeks on the scaffolds in chondrogenic differentiation medium. Cell proliferation on the scaffolds was determined by MTS assay and it was observed that all scaffolds supported cell proliferation. Immunostaining was carried out for morphological and differentiation analyses. Immunohistochemical analyses revealed that the cells attached onto the scaffolds and deposited cartilage‐specific extracellular matrix (ECM). Real‐time PCR was performed to determine the expression levels of cartilage‐specific genes. After 21 days of incubation in cartilage differentiation medium, expression of collagen type II increased only in the cells seeded onto PCL–PLLA blend scaffolds. Similarly, aggrecan expression was the highest on PCL–PLLA scaffolds after 3 weeks. These results suggest that all the scaffolds, and especially PCL–PLLA, were suitable for chondrogenic differentiation of HTGSCs. Copyright © 2015 John Wiley & Sons, Ltd.     

Fibrous biodegradable l‐alanine‐based scaffolds for vascular tissue engineering

In vascular tissue engineering, three‐dimensional (3D) biodegradable scaffolds play an important role in guiding seeded cells to produce matrix components by providing both mechanical and biological cues. The objective of this work was to fabricate fibrous biodegradable scaffolds from novel poly(ester amide)s (PEAs) derived from l ‐alanine by electrospinning, and to study the degradation profiles and its suitability for vascular tissue‐engineering applications. In view of this, l ‐alanine‐derived PEAs (dissolved in chloroform) were electrospun together with 18–30% w/w polycaprolactone (PCL) to improve spinnability. A minimum of 18% was required to effectively electrospin the solution while the upper value was set in order to limit the influence of PCL on the electrospun PEA fibres. Electrospun fibre mats with average fibre diameters of ~0.4 µm were obtained. Both fibre diameter and porosity increased with increasing PEA content and solution concentration. The degradation of a PEA fibre mat over a period of 28 days indicated that mass loss kinetics was linear, and no change in molecular weight was found, suggesting a surface erosion mechanism. Human coronary artery smooth muscle cells (HCASMCs) cultured for 7 days on the fibre mats showed significantly higher viability (p < 0.0001), suggesting that PEA scaffolds provided a better microenvironment for seeded cells compared with control PCL fibre mats of similar fibre diameter and porosity. Furthermore, elastin expression on the PEA fibre mats was significantly higher than the pure PEA discs and pure PCL fibre mat controls (p < 0.0001). These novel biodegradable PEA fibrous scaffolds could be strong candidates for vascular tissue‐engineering applications. Copyright © 2012 John Wiley & Sons, Ltd.     

Hydroxyapatite‐intertwined hybrid nanofibres for the mineralization of osteoblasts

Advances in tissue engineering have enabled the development of bioactive composite materials to generate biomimetic nanofibrous scaffolds for bone replacement therapies. Polymeric biocomposite nanofibrous scaffolds architecturally mimic the native extracellular matrix (ECM), delivering tremendous regenerative potential for bone tissue engineering. In the present study, biocompatible poly(l ‐lactic acid)‐co‐poly(ε‐caprolactone)–silk fibroin–hydroxyapatite–hyaluronic acid (PLACL–SF–HaP–HA) nanofibrous scaffolds were fabricated by electrospinning to mimic the native ECM. The developed nanofibrous scaffolds were characterized in terms of fibre morphology, functional group, hydrophilicity and mechanical strength, using SEM, FTIR, contact angle and tabletop tensile‐tester, respectively. The nanofibrous scaffolds showed a higher level of pore size and increased porosity of up to 95% for the exchange of nutrients and metabolic wastes. The fibre diameters obtained were in the range of around 255 ± 13.4–789 ± 22.41 nm. Osteoblasts cultured on PLACL–SF–HaP–HA showed a significantly (p < 0.001) higher level of proliferation (53%) and increased osteogenic differentiation and mineralization (63%) for the inclusion of bioactive molecules SF–HA. Energy‐dispersive X‐ray analysis (EDX) data proved that the presence of calcium and phosphorous in PLACL–SF–HaP–HA nanofibrous scaffolds was greater than in the other nanofibrous scaffolds with cultured osteoblasts. The obtained results for functionalized PLACL–SF–HaP–HA nanofibrous scaffolds proved them to be a potential biocomposite for bone tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.     

Modulation of chondrogenic differentiation of human mesenchymal stem cells in jellyfish collagen scaffolds by cell density and culture medium

Studies on tissue‐engineering approaches for the regeneration of traumatized cartilage focus increasingly on multipotent human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes. The present study applied porous scaffolds made of collagen from the jellyfish Rhopilema esculentum for the in vitro chondrogenic differentiation of hMSCs. Culture conditions in those scaffolds differ from conditions in high‐density pellet cultures, making a re‐examination of these data necessary. We systematically investigated the influence of seeding density, basic culture media [Dulbecco's modified Eagle's medium (DMEM), α‐minimum essential medium (α‐MEM)] with varying glucose content and supplementation with fetal calf serum (FCS) or bovine serum albumin (BSA) on the chondrogenic differentiation of hMSCs. Gene expression analyses of selected markers for chondrogenic differentiation and hypertrophic development were conducted. Furthermore, the production of cartilage extracellular matrix (ECM) was analysed by quantification of sulphated glycosaminoglycan and collagen type II contents. The strongest upregulation of chondrogenic markers, along with the highest ECM deposition was observed in scaffolds seeded with 2.4 × 10⁶ cells/cm³ after cultivation in high‐glucose DMEM and 0.125% BSA. Lower seeding densities compared to high‐density pellet cultures were sufficient to induce in vitro chondrogenic differentiation of hMSCs in collagen scaffolds, which reduces the amount of cells required for the seeding of scaffolds and thus the monolayer expansion period. Furthermore, examination of the impact of FCS and α‐MEM on chondrogenic MSC differentiation is an important prerequisite for the development of an osteochondral medium for simultaneous osteogenic and chondrogenic differentiation in biphasic scaffolds for osteochondral tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Targeted RGD nanoparticles for highly sensitive in vivo integrin receptor imaging

A new magnetic resonance imaging (MRI) contrast bearing RGD peptide is reported. In this study, ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles with various sizes were prepared. Particles sizes between 6 and 13 nm were tuned by varying the stirring rate. Remarkable negative contrast was observed because USPIO nanoparticles (13.1 ± 2.1 nm) generated high transversal relaxivity r₂ (188 ± 3 m m ^?1 s^?1) and saturation magnetization (94 emu g^?1 Fe). The USPIO nanoparticles were coated with PDA [2‐(pyridyldithio)‐ethylamine; PDA nanoparticles] containing functional polymer, which can be readily synthesized by Michael addition. The PDA nanoparticles were conjugated with RGD peptide (RGD nanoparticles) for targeting the specific site. The target specificity and high relaxivity allowed RGD nanoparticles to differentiate the expression level of integrin receptor on several cell lines and tumors (MCF‐7, A‐549, HT‐29 and HT‐1080) by in vitro and in vivo MR imaging. Importantly, a remarkable negative contrast (?51.3 ± 6.7%) was observed for in vivo MR imaging of MCF‐7 tumor. This result implies that the RGD nanoparticles that greatly enhance the MR imaging are highly sensitive for early stage tumor detection. Copyright © 2012 John Wiley & Sons, Ltd.     

Morpho‐functional response of the elbow extensor muscles to twelve‐week self‐perceived maximal resistance training

The aim of this study was to determine morphological and functional changes of the elbow extensor muscles in response to a 12‐week self‐perceived maximal resistance training (MRT). Twenty‐one healthy sedentary young men were engaged in elbow extensor training using isoacceleration dynamometry for 12 weeks with a frequency of five sessions per week (five sets of ten maximal voluntarily contractions, 1‐min rest period between each set). Prior to, at 6 weeks and after the training, a series of cross‐sectional magnetic resonance images of the upper arm were obtained and muscle volumes were calculated. Maximal and endurance strength increased (P<0·01) by 15% and 45% at 6 weeks, and by 29% and 70% after 12 weeks compared with baseline values, while fatigue rate of the elbow extensors decreased by 67%. The volume of triceps brachii increased in both arms (P<0·01) by 4% at 6 weeks, and by 8% after 12 weeks compared with baseline values (right arm – from 487·4 ± 72·8 cm³ to 505·8 ± 72·3 cm³ after 6 weeks and 525·3 ± 73·7 cm³ after 12 weeks; left arm – from 475·3 ± 79·1 cm³ to 493·2 ± 72·7 cm³ after 6 weeks and 511·3 ± 77·0 cm³ after 12 weeks). A high correlation was found between maximal muscle strength and muscle volume prior (r² = 0·62) and after (r² = 0·69) the training (P≤0·05). A self‐perceived MRT resulted in an increase in maximal and endurance strength. Morphological adaptation changes of triceps brachii as a result of 12‐week specific strength training can explain only up to 26% of strength gain.     

Imaging integrin alpha‐v‐beta‐3 expression in tumors with an 18F‐labeled dimeric RGD peptide

Integrin α_vβ₃ receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth. Many radiolabeled probes utilize the tight and specific association between the arginine–glycine–aspartatic acid (RGD) peptide and integrin α_vβ₃, but one main obstacle for any clinical application of these probes is the laborious multistep radiosynthesis of ¹⁸F. In this study, the dimeric RGD peptide, E‐[c(RGDfK)]₂, was conjugated with NODAGA and radiolabeled with ¹⁸F in a simple one‐pot process with a radiolabeling yield of 20%, the whole process lasting only 45 min. NODAGA‐E‐[c(RGDfK)]₂ labeled with ¹⁸F at a specific activity of 1.8 MBq nmol^?1 and a radiochemical purity of 100% could be achieved. The logP value of ¹⁸F‐labeled NODAGA‐E‐[c(RGDfK)]₂ was ?4.26 ± 0.02. In biodistribution studies, ¹⁸F‐NODAGA‐E‐[c(RGDfK)]₂ cleared rapidly from the blood with 0.03 ± 0.01 percentage injected dose per gram (%ID g^?1) in the blood at 2 h p.i., mainly via the kidneys, and showed good in vivo stability. Tumor uptake of ¹⁸F‐NODAGA‐E‐[c(RGDfK)]₂ (3.44 ± 0.20 %ID g^?1, 2 h p.i.) was significantly lower than that of reference compounds ⁶⁸Ga‐labeled NODAGA‐E‐[c(RGDfK)]₂ (6.26 ± 0.76 %ID g^?1; p <0.001) and ¹¹¹In‐labeled NODAGA‐E‐[c(RGDfK)]₂ (4.99 ± 0.64 %ID g^?1; p < 0.01). Co‐injection of an excess of unlabeled NODAGA‐E‐[c(RGDfK)]₂ along with ¹⁸F‐NODAGA‐E‐[c(RGDfK)]₂ resulted in significantly reduced radioactivity concentrations in the tumor (0.85 ± 0.13 %ID g^?1). The α_vβ₃ integrin‐expressing SK‐RC‐52 tumor could be successfully visualized by microPET with ¹⁸F‐labeled NODAGA‐E‐[c(RGDfK)]₂. In conclusion, NODAGA‐E‐[c(RGDfK)]₂ could be labeled rapidly with ¹⁸F using a direct aqueous, one‐pot method and it accumulated specifically in α_vβ₃ integrin‐expressing SK‐RC‐52 tumors, allowing for visualization by microPET. Copyright © 2013 John Wiley & Sons, Ltd.     

PET imaging of prostate tumors with 18F‐Al‐NOTA‐MATBBN

Overexpression of the gastrin‐releasing peptide receptor (GRPR) in prostate cancer provides a promising target for detection the disease. MATBBN is a new bombesin analog originating from the GRPR antagonists with a hydrophilic linker. In this study NOTA‐conjugated MATBBN was labeled by the Al¹⁸F method and the potential of ¹⁸F‐Al‐NOTA‐MATBBN for prostate tumor PET imaging was also evaluated. NOTA‐MATBBN was radiolabeled with ¹⁸F using Al¹⁸F complexes. Partition coefficient, in vitro stability and GRPR binding affinity were also determined. PET studies were performed with ¹⁸F‐Al‐NOTA‐MATBBN in PC‐3 tumor‐bearing mice. ¹⁸F‐Al‐NOTA‐MATBBN can be produced within 30 min with a decay‐corrected yield of 62.5 ± 2.1% and a radiochemical purity of >98%. The logP octanol–water value for the Al¹⁸F‐labeled BBN analog was ?2.40 ± 0.07 and the radiotracer was stable in phosphate‐buffered saline and human serum for 2 h. The IC₅₀ values of displacement for the ¹⁸F‐Al‐NOTA‐MATBBN with MATBBN was 126.9 ± 2.75 nm . The PC‐3 tumors were clearly visible with high contrast after injection of the labeled peptide. At 60 min post‐injection, the tumor uptakes for ¹⁸F‐Al‐NOTA‐MATBBN and ¹⁸F‐FDG were 4.59 ± 0.43 and 1.98 ± 0.35% injected dose/g, and tumor to muscle uptake radios for two tracers were 6.77 ± 1.10 and 1.78 ± 0.32, respectively. Dynamic PET revealed that ¹⁸F‐Al‐NOTA‐MATBBN was excreted mainly through the kidneys. GRPR‐binding specificity was also demonstrated by reduced tumor uptake of ¹⁸F‐Al‐NOTA‐MATBBN after coinjection with excess unlabeled MATBBN peptide at 1 h post‐injection. NOTA‐ MATBBN could be labeled rapidly with ¹⁸F using one step method. ¹⁸F‐Al‐NOTA‐MATBBN may be a promising PET imaging agent for prostate cancer. Copyright © 2014 John Wiley & Sons, Ltd.     

Syntheses and preliminary evaluation of [18F]AlF‐NOTA‐G‐TMTP1 for PET imaging of high aggressive hepatocellular carcinoma

The goal of this study is to evaluate a new ¹⁸F‐labeled imaging agent for diagnosing high metastatic (aggressive) hepatocellular carcinoma using positron emission tomography (PET). The new ¹⁸F‐labeled imaging agent [¹⁸F]AlF‐NOTA‐G‐TMTP1 was synthesized and radiolabeled with ¹⁸F using NOTA‐AlF chelation method. The tumor‐targeting characteristics of [¹⁸F]AlF‐NOTA‐G‐TMTP1 was assessed in HepG2, SMCC‐7721, HCC97L and HCCLM3 xenografts. The total synthesis time was about 20 min with radiochemical yield of 25 ± 6%. The specific activity was about 11.1–14.8 GBq/µmol at the end of synthesis based on the amount of peptide used and the amount of radioactivity trapped on the C₁₈ column. The log P value of [¹⁸F]AlF‐NOTA‐G‐TMTP1 was ‐3.166 ± 0.022. [¹⁸F]AlF‐NOTA‐G‐TMTP1 accumulated in SMCC‐7721 and HCCLM3 tumors (high metastatic potential) in vivo and result in tumor/muscle (T/M) ratios of 4.5 ± 0.3 and 4.7 ± 0.2 (n = 4) as measured by PET at 40 min post‐injection (p.i.). Meanwhile, the tumor/muscle (T/M) ratios of HepG2 and HCC97L tumors (low metastatic potential) were1.6 ± 0.3 and 1.8 ± 0.4. The tumor uptake of [¹⁸F]AlF‐NOTA‐G‐TMTP1 could be inhibited 61.9% and 57.6% by unlabeled G‐TMTP1 in SMCC‐7721 and HCCLM3 xenografts at 40 min p.i., respectively. Furthermore, [¹⁸F]AlF‐NOTA‐G‐TMTP1 showed pretty low activity in the liver and intestines in all tumor bearing mice, such in vivo distribution pattern would be advantageous for the detection of hepatic carcinoma. Overall, [¹⁸F]AlF‐NOTA‐G‐TMTP1 may specifically target high metastatic or/and aggressive hepatocellular carcinoma with low background activity and, therefore, holds the potential to be used as an imaging agent for detecting tumor lesions within the liver area. Copyright © 2016 John Wiley & Sons, Ltd.     

Additive manufacturing of poly[(R)‐3‐hydroxybutyrate‐co‐(R)‐3‐hydroxyhexanoate] scaffolds for engineered bone development

A wide range of poly(hydroxyalkanoate)s (PHAs), a class of biodegradable polyesters produced by various bacteria grown under unbalanced conditions, have been proposed for the fabrication of tissue‐engineering scaffolds. In this study, the manufacture of poly[(R)‐3‐hydroxybutyrate‐co‐(R)‐3‐hydroxyhexanoate] (or PHBHHx) scaffolds, by means of an additive manufacturing technique based on a computer‐controlled wet‐spinning system, was investigated. By optimizing the processing parameters, three‐dimensional scaffolds with different internal architectures were fabricated, based on a layer‐by‐layer approach. The resulting scaffolds were characterized by scanning electron microscopy, which showed good control over the fibre alignment and a fully interconnected porous network, with porosity in the range 79–88%, fibre diameter 47–76 µm and pore size 123–789 µm. Moreover, the resulting fibres presented an internal porosity connected to the external fibre surface as a consequence of the phase‐inversion process governing the solidification of the polymer solution. Scaffold compressive modulus and yield stress and strain could be varied in a certain range by changing the architectural parameters. Cell‐culture experiments employing the MC3T3‐E1 murine pre‐osteoblast cell line showed good cell proliferation after 21 days of culture. The PHBHHx scaffolds demonstrated promising results in terms of cell differentiation towards an osteoblast phenotype. Copyright © 2014 John Wiley & Sons, Ltd.     

Mechanotransduction in small intestinal submucosa scaffolds: fabrication parameters potentially modulate the shear‐induced expression of PECAM‐1 and eNOS

In small intestinal submucosa (SIS) scaffolds for functional tissue engineering, the impact of scaffold fabrication parameters on cellular response and tissue regeneration may relate to the mechanotransductory properties of the final arrangement of collagen fibres. We previously proved that two fabrication parameters, (a) preservation (P) or removal (R) of a dense collagen layer present in SIS, and (b) SIS in a final dehydrated (D) or hydrated (H) state, have an effect on the micromechanical environment of SIS. In a continuation of our studies, we herein hypothesized that these fabrication parameters also modulate early mechanotransduction in cells populating the scaffold. Mechanotransduction was investigated by seeding human umbilical vein endothelial cells (HUVECs) on scaffolds, exposing them to pulsatile shear stress (12 ± 4 dyne/cm²) for 1 h (n = 5) in a cone‐and‐plate shear system, and evaluating the expression of the mechanosensitive genes Pecam1 and Enos by immunofluorescence and qPCR. Expression of mechanosensitive genes was highest in PD grafts, followed by PH and RH grafts. The RD group had similar expression to that of unsheared control cells, suggesting that the RD combination potentially reduced mechanotransduction of shear to cells. We concluded that the two fabrication parameters studied, which modify SIS micromechanics, also potentially modulated the early shear‐induced expression of mechanosensitive genes in seeded HUVECs. Our findings suggest that fabrication parameters influence the outcome of SIS as a therapeutic scaffold. Copyright © 2015 John Wiley & Sons, Ltd.     

Osteogenesis of adipose‐derived stem cells on polycaprolactone–β‐tricalcium phosphate scaffold fabricated via selective laser sintering and surface coating with collagen type I

The current study aimed to fabricate three‐dimensional (3D) polycaprolactone (PCL), polycaprolactone and β‐tricalcium phosphate (PCL–TCP) scaffolds via a selective laser‐sintering technique (SLS). Collagen type I was further coated onto PCL–TCP scaffolds to form PCL–TCP–COL scaffolds. The physical characters of these three scaffolds were analysed. The osteogenic potential of porcine adipose‐derived stem cells (pASCs) was compared among these three scaffolds in order to find an optimal scaffold for bone tissue engineering. The experimental results showed no significant differences in pore size and porosity among the three scaffolds; the porosity was ca. 75–77% and the pore size was ca. 300–500 µm in all three. The compressive modulus was increased from 6.77 ± 0.19 to 13.66 ± 0.19 MPa by adding 30% β‐TCP into a 70% PCL scaffold. No significant increase of mechanical strength was found by surface‐coating with collagen type I. Hydrophilicity and swelling ratios showed statistical elevation (p < 0.05) after collagen type I was coated onto the PCL–TCP scaffolds. The in vitro study demonstrated that pASCs had the best osteogenic differentiation on PCL–TCP–COL group scaffolds, due to the highest ALP activity, osteocalcin mRNA expression and mineralization. A nude mice experiment showed better woven bone and vascular tissue formation in the PCL–TCP–COL group than in the PCL group. In conclusion, the study demonstrated the ability to fabricate 3D, porous PCL–TCP composite scaffolds (PCL:TCP = 70:30 by weight) via an in‐house‐built SLS technique. In addition, the osteogenic ability of pASCs was found to be enhanced by coating COL onto the PCL–TCP scaffolds, both in vitro and in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Dual‐isotope 111In/177Lu SPECT imaging as a tool in molecular imaging tracer design

The synthesis, design and subsequent pre‐clinical testing of new molecular imaging tracers are topic of extensive research in healthcare. Quantitative dual‐isotope SPECT imaging is proposed here as a tool in the design and validation of such tracers, as it can be used to quantify and compare the biodistribution of a specific ligand and its nonspecific control ligand, labeled with two different radionuclides, in the same animal. Since the biodistribution results are not blurred by experimental or physiological inter‐animal variations, this approach allows determination of the ligand's net targeting effect. However, dual‐isotope quantification is complicated by crosstalk between the two radionuclides used and the radionuclides should not influence the biodistribution of the tracer. Here, we developed a quantitative dual‐isotope SPECT protocol using combined ¹¹¹Indium and ¹⁷⁷Lutetium and tested this tool for a well‐known angiogenesis‐specific ligand (cRGD peptide) in comparison to a potential nonspecific control (cRAD peptide). Dual‐isotope SPECT imaging of the peptides showed a similar organ and tumor uptake to single‐isotope studies (cRGDfK–DOTA, 1.5 ± 0.8%ID cm^?3; cRADfK–DOTA, 0.2 ± 0.1%ID cm^?3), but with higher statistical relevance (p‐value 0.007, n = 8). This demonstrated that, for the same relevance, seven animals were required in case of a single‐isotope test design as compared with only three animals when a dual‐isotope test was used. Interchanging radionuclides did not influence the biodistribution of the peptides. Dual‐isotope SPECT after simultaneous injection of ¹¹¹In and ¹⁷⁷Lu‐labeled cRGD and cRAD was shown to be a valuable method for paired testing of the in vivo target specificity of ligands in molecular imaging tracer design. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthetic poly(amino acid) hydrogels with incorporated cell‐adhesion peptides for tissue engineering

Preparation of soft poly(amino acid) hydrogels containing biomimetic cell‐adhesive peptides was investigated. Covalently crosslinked gels were formed by radical co‐polymerization of methacryloylated macromonomer poly[N⁵‐(2‐hydroxyethyl)‐L ‐glutamine‐stat‐L ‐alanine‐stat‐methacryloyllysine] with 2‐hydroxyethyl methacrylate (HEMA) as minor co‐monomer. Hydrogels carrying biomimetic peptides were prepared by using methacryloylated peptides, such as methacryloyl–GGGRGDSG–OH and methacryloyl–GGGYIGSR–OH, as additional monomers in the polymerization mixture. Mechanical stability and swelling in water of the hydrogels obtained for different solid:water and polypeptide:HEMA ratios were evaluated. The microporosity of gels (5–20 µm), dependent on the polyHEMA phase separation in water, was followed by low‐vacuum SEM. The effect of biomimetic modification of hydrogels with RGDS and YIGSR peptides on the seeding efficiency of porcine mesenchymal stem cells (MSCs) was studied in vitro. While unmodified hydrogels showed very low cell adhesion, due to their highly hydrophilic nature, the incorporation of adhesive peptides significantly improved the adhesion and viability of seeded cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Decellularization of bovine small intestinal submucosa and its use for the healing of a critical‐sized full‐thickness skin defect,alone and in combination with stem cells,in a small rodent model

In this study, we initially described an efficient decellularization protocol for bovine‐derived small intestinal submucosa (bSIS), involving freeze–thaw cycles, acid/base treatment and alcohol and buffer systems. We compared the efficacy of our protocol to some previously established ones, based on DNA content and SEM and histochemical analyses. DNA content was reduced by ~89.4%, significantly higher than compared protocols. The sulphated GAG content of the remaining interconnected fibrous structure was 5.738 ± 0.207 µg/mg (55% retained). An in vitro study was performed to evaluate whether rat bone marrow mesenchymal stem cells (MSCs) could attach and survive on bSIS membranes. Our findings revealed that MSCs can preserve their viability and proliferate on bSIS for > 2 weeks in culture. We conducted in vivo applications for the treatment of an experimental rat model of critical sized (7 cm²) full‐thickness skin defect. The wound models treated with either MSCs‐seeded (1.5 × 10⁶ cells/cm²) or non‐seeded bSIS membranes were completely closed by week 7 without significant differences in closure time; on the other hand, the open wound control was closed at ~47% at this time point. Immunohistopathology results revealed that the group which received MSCs‐seeded bSIS had less scarring at the end of the healing process and was in further stages of appendage formation in comparison with the non‐seeded bSIS group. Copyright © 2015 John Wiley & Sons, Ltd.     

Preliminary evaluation of novel 68Ga‐DOTAVAP‐PEG‐P2 peptide targeting vascular adhesion protein‐1

Introduction: Expression of vascular adhesion protein‐1 (VAP‐1) is induced at the sites of inflammation where extravasation of leukocytes from blood to the peripheral tissue occurs. VAP‐1 is a potential target for anti‐inflammatory therapy and for in vivo imaging of inflammation. Purpose of this study was to preliminarily evaluate a novel VAP‐1‐targeting peptide as a potential PET imaging agent. Methods: Cyclic 17‐amino‐acid peptide selected from phage display libraries was 1,4,7,10‐tetraazacyclododecane‐N,N′,N′′,N′′′‐tetraacetic acid (DOTA) conjugated via 8‐amino‐3,6‐diooxaoctanoyl linker (polyethylene glycol, PEG derivative) and labelled with ⁶⁸Ga (⁶⁸Ga‐DOTAVAP‐PEG‐P2). In vitro stability of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was determined in saline, rat plasma and human plasma by radio‐HLPC. Lipophilicity was measured by calculating octanol‐water partition coefficient (logP). Whole‐body distribution kinetics and stability after intravenous injection in healthy rats was studied in vivo by PET imaging, ex vivo by measuring radioactivity of excised tissues, and by radio‐HPLC. Results: In vitro the ⁶⁸Ga‐DOTAVAP‐PEG‐P2 remained stable >4 h in saline and rat plasma, but degraded slowly in human plasma after 2 h of incubation. The logP value of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was ?1·3. In rats, ⁶⁸Ga‐radioactivity cleared rapidly from blood circulation and excreted quickly in urine. At 120 min after injection the fraction of intact ⁶⁸Ga‐DOTAVAP‐PEG‐P2 were 77 ± 6·0% and 99 ± 1·0% in rat plasma and urine, respectively. Conclusions: These basic and essential in vitro and in vivo studies of the new VAP‐1 targeting peptide revealed promising properties for an imaging agent. Further investigations to clarify in vivo VAP‐1 targeting are warranted.     

Human platelet antigen polymorphisms (HPA‐1, ‐2, ‐3, ‐4, ‐5 and ‐15) in major ethnic groups of Pakistan

Objectives: Gene frequencies of human platelet antigens (HPA) determine the magnitude of platelet immunological disorders like neonatal alloimmune thrombocytopenia, platelet refractoriness and ease of availability of particular HPA‐typed platelet donors in a given community. Background: However, the pattern of HPA in Pakistani population is not known. Aim: The aim of present study was to determine the gene frequencies of HPA (HPA‐1 to ‐5 and ‐15) in individuals belonging to major ethnic groups and castes of Pakistani population. Materials and Methods: HPA genotyping was done in 593 individuals belonging to all ethnic groups of Pakistan, by polymerase chain reaction‐sequence specific primers with detection on polyacrylamide electrophoresis. Results: The gene frequencies of the ‘a’ and ‘b’ alleles of HPA‐1 to ‐5 and ‐15 in Pakistanis were as follows: HPA‐1a/b, 0·885/0·115; HPA‐2a/b, 0·92/0·08; HPA‐3a/b, 0·69/0·31; HPA‐4a/b, 1/0; HPA‐5a/b, 0·9/0·1; HPA‐15a/b, 0·59/0·41. Except for significant difference regarding gene frequency of HPA‐3 between Pathans and Sindhis, there was no significant difference of HPA‐1 to ‐5 and ‐15 between major ethnic groups of Pakistan. The estimated mismatch probability regarding platelet antigens 1–5 and 15 in Pakistanis, after transfusion of random donor platelets, is from 14 to 37%. The expected incidence of neonatal alloimmune thrombocytopenia due to anti‐HPA‐1a in Pakistani pregnant females is < 1 of 1000 pregnancies and 8–12 of 1000 in case of anti‐HPA‐5b. Homozygosity of HPA‐1b, ‐2b and ‐5b genotypes ranged from 1 to 2% in the Pakistani population, whereas homozygosity of HPA‐3b and ‐15b was 11 and 18%. Conclusions: There is a need to establish donor registries typed for HPA in the transfusion centres of the country.     

Magnesium calcium phosphate/β‐tricalcium phosphate incorporation into gelatin scaffold: an in vitro comparative study

Gelatin scaffolds incorporating or not 50 wt% of magnesium calcium phosphate (MCP) or β‐tricalcium phosphate (βTCP) were prepared and the in vitro osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs) in the scaffolds was investigated. The pore sizes of the scaffolds were in the range 123.8 ± 47.2–153 ± 60.72 µm in diameter, while the porosity was 33.3 ± 2–44.9 ± 3.4%. The compression modulus of the sponges was about 2.04–2.24 mPa. There was no significant difference among groups regarding the physical and mechanical properties. When seeded into the sponges by an agitation method, the MSCs were distributed throughout the scaffold. Higher MSC proliferation was observed for scaffolds incorporating minerals. Following the incubation of MSCs in scaffolds incorporating MCP, the alkaline phosphatase activity was significantly higher at weeks 2, 3 and 4 in comparison with other scaffolds; however, the osteocalcin levels of MSCs did not show significant differences among groups. These findings indicate that MSCs seeded in scaffolds incorporating MCP showed significantly superior biological results in terms of proliferation and osteogenic differentiation in comparison with other scaffold types. Copyright © 2012 John Wiley & Sons, Ltd.     

Directional migration of endothelial cells towards angiogenesis using polymer fibres in a 3D co‐culture system

Development of an in vitro prevascularized scaffold is of great importance to produce vascularization in tissue‐engineered devices and for other clinical purposes. To this aim, polymer fibres covered with human umbilical vein endothelial cells (HUVECs) were used to induce directional ‘angiogenesis’ in a 3D co‐culture system. Gelatin or RGD peptides were immobilized on surface‐modified polymer fibres [100 µm diameter poly(ethylene terephthalate) monofilaments] via N‐hepthylamine plasma polymer and carboxy‐methyl‐dextran interlayers. Fibres fully covered with HUVECs were then embedded in a fibrin gel, following a parallel alignment pattern, in the presence of fibroblasts. Tube‐like structures occurred along the fibres and a network was formed between neighbouring fibres. These events were promoted with increased incubation times. Biomolecule‐grafted fibres created a guidance pathway that facilitated coated endothelial cells to form lumens and, from them, sprouting processes. However, there were no significant differences between the different surface modifications on fibres in terms of promoting tube‐like structures. Thus, different stages of angiogenesis can be initiated and guided using HUVECs precovered polymer fibres embedded in a soft supportive matrix, such as fibrin, which can be further applied to the development of in vitro prevascularized tissue‐engineered scaffolds. Copyright © 2010 John Wiley & Sons, Ltd.     

Nanofibrous nerve conduit‐enhanced peripheral nerve regeneration

Fibre structures represent a potential class of materials for the formation of synthetic nerve conduits due to their biomimicking architecture. Although the advantages of fibres in enhancing nerve regeneration have been demonstrated, in vivo evaluation of fibre size effect on nerve regeneration remains limited. In this study, we analyzed the effects of fibre diameter of electrospun conduits on peripheral nerve regeneration across a 15‐mm critical defect gap in a rat sciatic nerve injury model. By using an electrospinning technique, fibrous conduits comprised of aligned electrospun poly (ε‐caprolactone) (PCL) microfibers (981 ± 83 nm, Microfiber) or nanofibers (251 ± 32 nm, Nanofiber) were obtained. At three months post implantation, axons regenerated across the defect gap in all animals that received fibrous conduits. In contrast, complete nerve regeneration was not observed in the control group that received empty, non‐porous PCL film conduits (Film). Nanofiber conduits resulted in significantly higher total number of myelinated axons and thicker myelin sheaths compared to Microfiber and Film conduits. Retrograde labeling revealed a significant increase in number of regenerated dorsal root ganglion sensory neurons in the presence of Nanofiber conduits (1.93 ± 0.71 × 10³ vs. 0.98 ± 0.30 × 10³ in Microfiber, p < 0.01). In addition, the compound muscle action potential (CMAP) amplitudes were higher and distal motor latency values were lower in the Nanofiber conduit group compared to the Microfiber group. This study demonstrated the impact of fibre size on peripheral nerve regeneration. These results could provide useful insights for future nerve guide designs. Copyright © 2012 John Wiley & Sons, Ltd.